Germicide



Patented Sept. 17, 1935 GERMICIDE Lyle AQWeed, Iowa City, Iowa, assignortoThe Hamilton Laboratories, Inc., Hamilton, Ohio, a corporation ofDelaware No Drawing. Application November 19, 1934,

' Serial No. 753,786

9 Claims. (Cl. 167-71) This invention relates to germicides suitable forinternal and external medication and to methods of destroying, in thepresence of living tissue, micro-organisms which are pathogenic to manor higher animals but without harming the tissue.

The germicidal pharmaceutical preparations embraced in the presentinvention are organic mercury compounds in .which the mercury atom 10 itattached by one valence to a carbon atom of a benzene ring and by theother to an atom or radical which forms an anion when the compound isdissolved in water. These compounds ionize 'in aqueous solution in sucha manner that the mercury atom forms a partof the positively charged ionor cation. It is possible that their unusual effectiveness againstbacteria is due to this fact, since bacteria carry a negative chargewhich promotes the adsorption of cations. Generically, these organicmercury compounds may be represented by theformula, R-Hg-X, wherein Rrepresents a phenyl radical in which any or none of the hydrogen atomsare substituted by non-metallic elements or radicals, which when presentin the said R-Hg-X compound will not react with either alkalies or acidsto form salts,

' and wherein X represents an-element or radical which exists as ananionwhen the compound is dissolved in water. Y 0 In case the benzenering has. no othersubstitutions, the compounds is a phenylmercuriccompound havingthe formula CsHsHgX, X being an anion; In thisapplication, unless otherwise stated, the term -"pheny1mercuric compound35 means CcHsHgX, X being an anion, and the term phenylmercuric radical.or phenylmercuric ion" means CsH5Hg+. Certain germicidal preparations ofthis type are described in my copending applications, Serial No.625,657, filed July 0 29, 1932, and Serial No. 736,105, filed July-19,1934,

of which applications this is a continuation-inpart. I have also foundthat certain substituted phenylmercuric compounds have great usefulnessas germicidal and antiseptic preparations. By

45 substituted phenylmercuric compounds I mean phenylmercuric compoundsin which one or more of the remaining hydrogen atoms in the benzene ringis replaced by another element or group. Amongthe groups that may beadvantageously substituted in the benzene ring are halogen, nitro,methyl and other hydrocarbon groups. Salt forming or solubilizinggroups, such as the hydroxyl, carboxyl, sulfonic, aminoand dimethylaminogroups, either in the free state or as salts, W attacheddirectly to thebenzene nucleus, greatly change the character of the compounds. Suchsolubilizing. groups are not included as substituents in thephenylmercuric radical of the cornpounds of the present invention. It ishowever,

is included, there being no salt forming groups as a substituent in thephenylmercuric radical. 1 Also, compounds having more than one hydrogenatom replaced by the same or by different non-salt forming groups in thephenylmercuric radical give satisfactory pharmaceutical preparations;for example, chlornitrophenylm'ercuric acetate. Phenylmercuric acetatemay. be conveniently made by mercurating benzene by-means oi mercuricacetate in the presence of glacial acetic acid. O-nitro and halogenphenylmercuricace- 25 tates may be made in a similar manner fromnitrobenzene or halogen benzene,- respectively. P-tolylmercuric chloridemay be. prepared from sodium p-toluene sulfinate and mercuric chloride(Organic Syntheses, Vol. III, page 99, H. T. Clarke, Editor, John Wiley8; Sons 1923"). me and p-nitrophenylmercuric chloride may be prepared inan analogous wayfrom mand p-nitrobenzene suliinic acids, respectively.The Grignard synthesis may also be employed for the preparation of thecompounds.

The acetate or chloride may be replaced in these compounds by otheranions by double decomposition; for example, phenylmercuric ace tate maybe reacted with sodium salicylate' in water solution, giving aprecipitate oi the less soluble phenylmercuric salicylate. Alsophenylmercuric chloride may be reacted with silver nitrate in alcoholicsolutions, giving a solution of phenylmercuric nitrate and a precipitateoi. 311- ver chloride as a by-product.

As a class, phenylmercuric compounds and substituted phenylmercuriccompounds are fairly insoluble in water. Thus, phenylmercuric acetate,which is one of the'most soluble, dissolves to the extent 01' about 0.5party in 100 parts cold water and of about 4 per cent in boiling water.'Ph'enyb mercuric chloride is soluble in about 20,000 parts a of water,the bromide to a less extentrand the iodide still less, P-tolylmercuricchloride 11501- hydroxide, and consequently, under these conditions, theapparent solubility of any salt is really the solubility of thehydroxide.

Phenylmercuric compounds of the class described, those carryingan anion'linked to a benzene nucleus through a mercury atom, are relativelystable in the animal system. In the medical use of these salts, theirlimited solubility is an advantage, since it enables automaticpreparation of solutions which are eflectively lethal against bacteriabut are not strong enough to be irritant.

I have found that the precipitated product resulting when a saturatedsolution of phenylmercuric acetate is reacted with a strong aqueoussolution of potassium nitrate, has a solubility of an order which makesit quite suitable for the present purpose. The product resulting fromthis reaction corresponds in its reactions, and the analysis of theproduct conforms to, a basic nitrate having the formula (csHsHg)zOI-LNOa, al-

- though possibly the composition is an equimolecular mixture ofphenylmercuric nitrate and phenylmercuric hydroxide. It iscrystallizable' and the crystals appear homogeneous, making it probablethat it is a compound. This product melts with decomposition attemperatures in the neighborhood of l'76-186 C. This compound is solublein about 1200 parts of water, and this solution, like many of thephenylmercuric salts, is colorless,:almost tasteless and non-corrosiveto the heavy metals. It is somewhat more soluble in alcohol, glycerin,diethylene glycol and acetone.

Addition of one mol of nitric acid to an acetone solution of one mol ofbasic phenylmercuric nitrate gives the normal phenylmercuric nitrate,which can be recrystallized from water to reproduce the basic nitrate.

- The choice of the particular phenylmercuric or substitutedphenylmercuric compound empolyed in any given case depends on thecircumstances.

Each has specific advantages in particular relations and they arepowerful germicides, relatively non-toxic to higher animals.

present investigation has included the preparation of a number ofphenylmercuric compounds, including in'addition to those'alreadymentioned the propionate, butyrate, pic rate, basic picrate, sulfate,carbonate, phosphate, and borate. The centesimal composition has notbeen determined in all cases and it is possible that some of thesecompounds have in fact been basic salts. In general they may be preparedby mixing a hot concentrated aqueous .solution of. a relatively. solublephenylmercuric compound of a relastrong solution of a salt containingthe desired anion, and allowing the less soluble phenylmercuric salt toseparate out, then washing and recrystallizing. Non-aqueous solvents mayoften be employed. Thus, phenylmercuric picrate may be prepared bymixing together 22.9 g. picric acid and33.6 g. phenylmercuric acetate in200cc. hot benzene until a solution is obtained, filtering, cooling, andallowing the phenylmercuric picrate to settle out, washing andrecrystallizing'from benzene. If the reaction is carried out in water,the basic picrate is formed. It is, however, important that thecompounds be of a high degree of purity. The presence of by-productsobtained in the nianuf acture has been'iound to cause a markeddiminution in 2,o14,eve

germicidal activity, accompanied by increased toxicity toward the higheranimals. These byproducts, or side products, do not have the particularmolecular structure which I have found useful. I, r

I believe these compounds to be the most eilective germicides andantlseptics now known to combat bacteria, fungi, and yeasts pathogenicto human beings. To a less extent, they are elective against protozoa.10 Using the Reddish technic, the following group of organisms waskilled in five minutes by basic phenylmercuric nitrate andphenylmercuric chloride in dilutions, of from 1:50,000 to as high asl:350,000:-Streptococcus hemolyticus, B typhosus, B pyocyane'us,Staphylococcus aureus, Proteus vulgaris, B mucosus capsulatus and Bcoli. Using the same procedure, p-tolylmercuric chloride killed 13 coliat a dilution of 1:50.000.

Cultures of Staphylococcus aureus were grown 20 on agar slants for 36hours and then made into a suspension in physiological saline solutionand a standard amount of this suspension was added to a standard amountof the basic phenylmercurio nitrate. Transfers were made to inoculation8 tubes of broth after 3 minutes, and the. tubes were then incubated 48hours. At a concentration of 1:75,000, the organisms were killed,whereas the control showed 2x10 living organisms per cubic centimeter.l-

With pathogenic fungi includingpathogenic yeasts, basic phenylmercuricnitrate shows 1'2? markable results. For instance, Trichophytonnodoformans after 28 'days' incubation, showed no growth in the presenceof the compound at a concentration of'1:l25,000. The compounds embracedby' this invention are strikingly effective in inhibiting bacterialgrowth. Thus basic phenylmercuric nitrate-was found to inhibit thegrowth of B coli at a dilution 40- of 1 part in 420,000 and ofStaphylococcus aureus at a dilution of 1 part in 12,000,000. Similarly,basic phenylmercuric picrate prevented growth of B coli at. a dilutionof l:800,000, and of Staphylococcus aureus at a dilution'of l:8.400,000.45.

salicylate at a dilution of 1:20,000 in olive oiilii prevented B coligrowth over an area of y; inch, and at a dilution of l:6.400,000preventedgrowth of Staphylococcus aureus over an area of 56 inch. Acrystal of phenylmercuric bromide applied to the agar plate left a clearspace of inch in the'fl' case of B coli and 1% inches withStaphylococcil aureus. The germicidal effectiveness of phenylmercuriccompounds against these organisms is only slightly reduced in thepresence of animal tissues.

Phenylmercuric compounds are relatively nontoxic to higher animals. Thusrats, mice and guinea pigs were given saturated solutions of basicphenylmercuric nitrate as their only source of drinking water forperiods of from 7 to 18 days; 70 0f the thirty-seven animals subjectedto this extremely severe test, twenty-seven appeared normal at the endof the period. Successive intravenous injections of 5 cc. of a saturatedsolution of basic phenylmercuric 111- 70.

G Rabbits tolerate 7.5 mg. basic phenylmercuric trate were administeredto rabbits on the third and fifth day of a 14 day test. The animalsremained normal in every respect throughout the 14 days they were underobservation.'

nitrate per kilo body weight intraperitoneally and about the same doseof p-tolylmercuric chloride.

.Phenylinercuric chloride in mg. doses has,

been administered orally to man thrice daily for repeated periods ofweeks without any untoward symptoms. In another test, 250 cc. of thesaturated aqueous solution of the basic phenylmercuric nitrate takenorally by a man resulted in no signs of mercurypoisoning.

Phe'nyimercuric compounds may be used in the form of aqueous solutions,with or without the addition of glycerimalcohol orv other, agents. Inthis form, they are valuable antisepties'for application to cuts, woundsor abrasions, or as mouth washes and douches. In a clinical study of 100cases involving infection of the vagina and cervix, all but tworesponded favorably to local application and douches of solutions ofbasic phenylmerciu'ic nitrate. The two excepted cases were infectionsbythe quite resistant protozoan, Tri- -chomonas vaginaies All gonorrhea]infections were cured.

The infection in these cases, as is usual, was characterized by livingorganisms which, except in the case of'Trichomonas, were fungi living inclose proximity to cells of an animal nature. On applying thesolution'to the surface, that is, bringing it in contact with both typesof cells, the fungi cells and the-cells of the living animal tissue, thetormer were selectively killed, while the animal cells were uninjured.There was but little irritation felt by the patient, and no irritationoia lasting nature was experienced. In other words, an application ofthe solution to vegetable microorganisms adjacent to animal cells killedthe former and did not aifect the latter.-

Basic phenylmercuric nitrate in olive oil is a 'particularly valuabletherapeutic agent in the treatment of fungus or other germ growths onthe skin. Ointments embodying basic phenyl- -mercuric nitrate. inan-oxycholesterin base have a similar usefulness as evidenced in theunusually successful treatment of tinea and yeast infections of theskin. The microorganisms causing the diseases were destroyed withoutharmful eifects on the patients.

Phenylmercuric compounds are likewise valuable in the treatment ofinternal infections, which cannot be reached by'topical medication, inwhich casesthey are administered by mouth, intravenous injection, etc.Colitis, septicemia, pneumonia, cystitis,- biliary infections and otherdiseases,

inwhich it is diiilcult or dangerous to reach the foci'ofinfection,-have responded favorably to internal administration ofphenylmercuric compounds.

also, diseases which are considered to be-due to iilterable viruseshave'been successfully treated by the application ot phenylmercuriccompounds.

As stated, it is important that 'theoompounds be of a high degree of.purity. As an illustration of amethod of manufacturing a pharmaceuticalpreparation within the purview of my invention, one.,pou nd of mercuricacetate is added to one of hot glacial acetic acid introduced into aflask containing 900'cc. of thiophenef benzene. The mixture isautoclaved for four hours at a pressureofapprozimately 8 pounds.

first,

action mixture cooledto'roomftemperaturm to C. The precipitatewhichisformed on cooling is separated from the liquid portion by filtration.The filtrate is evaporated to complete dryness. The resulting residue isthen introduced into boiling water-1n Wliich-it Is largelQ soluble. Theinsoluble portion is separated by filtration from the hot water solutionand-the filtrate allowed to cool to" room*-"'tempeiatire.

Crystals of phenylmercur-ic acetate ferm ing and are separated byilltratiorrI The -phenyl'i- .mercuric acetate so obtained-'is=redissowedin water and an excess of sodium 'nitrate is 'sdded.

A separation of basic pl'ienyln-iercurio r'litrate' ooicurs, which isremoved by 'filtrationxt A s-simian l'ii of this basic phenylmercuricnitrate, 1 part in 1250 parts of water, is a suitable pharmaceuticalpreparation.

In event phenylmercuric chloride is made, the phenylmercuric. acetateobtained as above de- 20 scribed is treated with sodium chloride inaqueous solution.

What I claim is:

1. The method of treating pathogenic germs while they are in contactwith tissue of. living '25 higher animals for the purpose of renderingthe germs innocuous and without harming the animal, which comprisescontacting said germs in situ with asolution of a suitable concentrationof an organic formula wherein X is an element or radical which exists asan anion when the compound is dissolved in water.

4. The method. of claim 1 wherein the organic mercury compound is basicphenylmercuric nitrate.

5. A germicidal preparation for use in suitable concentration in contactwith tissue of'a' living human being or other higher. animal for thepurpose of combating the attack of pathogenic micro-organisms on saidanimal, without harming the animal, comprising an organic mercurycompound having the formula wherein It represents a phenyl radicalcarrying no substituent'gr'oups which will react with either 05 alkaliesor acidsto form salts and wherein x represents an element or radicalwhich exists as an anion when the compound is dissolved in water.

'6; The germicidal preparation of claim 5.in which R represents asubstituted phenyl radical in which the substituents are selected fromthe .cla'ss consisting of halogen, nitro, and hydrocarbon.

mercury compound having the I. The germicidal preparation of claim 5where- 86 I 4: in said organic mercuric compound is a phenyl mercurycompound having the formula Cal-IsHgX wherein X is an element or radicalwhich exists as an anion when the compound is dissolved in.

water. I

8.. The germicidal preparation of claim 5 where- 10 in said organicmercuric compound is basic phenylmercuric nitrate.

9. As a germicidal preparation for use in contact with tissue of livinghuman beings or other higher animals for the purpose of combating the 15attack oi pathogenic micro-organisms on said

